The Na+ sensitivity of whole brain membrane Na+,K+-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha1, low affinity for ouabain; alpha2, high affinity; and alpha3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+, K+-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha1, exhibited high Na+ sensitivity [K(a) of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13]; the ouabain high-affinity isoform, alpha2, had two Na+ sensitivities, a high (K(a) of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (K(a) of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha3, was resolved by two processes and appears to have two Na+ sensitivities (apparent K(a) values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.
